In eukaryotic biology, the terms cilia and flagella both refer to highly organized membrane-bound, microtubule-based organelles that project from cells. Often the terms are used interchangeably, but, in this review, we use the term cilia throughout. Cilia are of interest to cell biologists in terms of their essential roles in diverse cell types, the mechanisms that lead to their formation and the features that distinguish motile from sensory functions. Cilia were first studied in unicellular organisms, such as algae and trypanosomes, or in sperm cells (reviewed in Gibbons, 1981). These systems facilitated ultrastructural characterization, analyses of motility, forward mutagenesis screens for ciliary defects and the identification of genes encoding ciliary proteins. Subsequent studies in mammalian cell lines provided further insight into cilia assembly and function (reviewed in Satir & Christensen, 2007). In the last decade, however, the field has been revolutionized by the advent of proteomics and of bioinformatic approaches to genome interrogation. Not only are comparative analyses now possible between diverse species, but organisms that have previously been experimentally intractable can now be included in analyses that aim to identify novel ciliary components and to characterize the evolutionary trajectory of cilia across the eukaryotes.
In particular, the new technologies are enabling comparative analyses of ciliogenesis in the Viridiplantae (green algae and land plants). Although the green alga Chlamydomonas reinhardtii has long been a workhorse in the cilia field (reviewed in Silflow & Lefebvre, 2001), studies of ciliated land plants have previously been limited by the paucity of experimentally tractable systems. It should be noted, however, that the iconic 9 + 2 microtubule arrangement in cilia (see paragraph below) was first observed in plant sperm (Manton & Clarke, 1951). In this review, we provide an overview of cilia form, function and loss in the land plants, and we use insights gained from comparative genomic studies to speculate on how cilia evolved within the Viridiplantae. To underpin this discussion, we first outline our current understanding of cilia diversity in other lineages – in terms of structure, assembly and function.
1. Basic ciliary structure
A canonical cilium consists of a microtubule axoneme that extends from a basal body (Fig. 1) (reviewed in Satir & Christensen, 2007). The basal body comprises two structurally distinct regions that differ in microtubule composition; the proximal end contains nine symmetrically arranged triplets of A, B and C microtubules, whereas the more distal transition zone comprises nine doublet microtubules. This difference arises because only the A and B microtubules extend into the transition zone. The canonical axoneme is a ring of nine outer microtubule doublets that are connected via nexin links, surrounding a central pair of singlet microtubules (known as the ‘9 + 2’ arrangement). Specialized inner- and outer-arm dynein motors are attached to the A microtubule of each doublet, and positioned in such a manner that the motor head domains are in close proximity to the B microtubule of the neighbouring doublet (Gibbons & Gibbons, 1973). Radial spokes attach the central pair microtubules to the surrounding doublets. In most species, bidirectional intraflagellar transport (IFT), which is based on the action of the specialized microtubule motors cytoplasmic dynein-2 and kinesin-2, carries cargo towards the distal tip of the axoneme and back (Kozminski et al., 1993; reviewed in Rosenbaum & Witman, 2002).
Figure 1. Microtubule structure of a eukaryotic cilium. Diagrammatic illustration of the canonical microtubule arrangement in the basal body (blue) and axoneme (green) of a eukaryotic cilium. Basal bodies are composed of two structurally distinct regions: a proximal region of nine triplet microtubules (A, B, C) and a transition zone of nine doublet microtubules (A, B). Axonemes are composed of nine doublet microtubules (A, B) and a microtubule central pair (CP).
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2. Diversity in cilia assembly
All cilia are formed by microtubule extension from basal bodies, but the process of ciliary assembly is variable across the eukaryotes. In many species, the basal body is formed from a triplet centriole that is embedded in the centrosome. In the slime mould Physarum flavicomum, when the amoeboid phase encounters water, the centriole pair moves from its juxtanuclear position, docks at the cell membrane and extends to form one long and one short cilium from the mature centriole and immature pro-centriole, respectively (Aldrich, 1968). When water is removed, the cilia are resorbed. This feature is also seen in other protists, such as amoeboflagellates that can switch between amoeboid and flagellate forms (Balamuth et al., 1983). In these cases, ciliogenesis is thus induced by environmental cues.
In contrast with the inductive formation seen in Physarum, cilia are always present in trypanosomes. In these organisms, basal bodies are never found in a centrosomal (centriolar) context, but exist as a pair made up of a mature basal body subtending the axoneme and an associated (immature) pro-basal body. During the cell cycle, the pro-basal body matures to form the new cilium and new pro-basal bodies form next to each of the original basal bodies. In this way, each daughter cell has a basal body with a cilium and a pro-basal body (reviewed in Ginger et al., 2008).
In animal cells, two distinct processes of basal body duplication and ciliogenesis operate depending on the cell type involved and developmental status. In most dividing cells, centrioles are produced in S phase by a process of templated duplication similar to that seen for the basal bodies of trypanosomes (Fig. 2a) (Cavalier-Smith, 1974; Dirksen, 1991; Quarmby & Parker, 2005). This duplication process involves the separation of existing centrioles and the formation of daughter centrioles orthogonally alongside the mature centrioles (reviewed in Cunha-Ferreira et al., 2009). Ciliogenesis is thus linked to the cell cycle – occurring (if at all) by the formation of a transient primary cilium in G1 or a more permanent cilium when the cell exits the cell cycle in G0. Importantly, this assembly process has an inherent number control system because, for each cilium in the mother cell, duplication and subsequent mitosis result in a centriole pair in each daughter cell. Notably, the widespread phylogenetic distribution of the templated pathway suggests that this mechanism was present in the last eukaryotic common ancestor (LECA) (Cavalier-Smith, 1974).
Figure 2. Basal body duplication pathways in mammalian cells. (a) Template duplication pathway in dividing cells. In G2/S, cilia are disassembled and centriole (red)/pro-centriole (blue) pairs are released from the membrane. During G1, the pro-centriole matures into a centriole and duplication then occurs to form two new pro-centrioles (grey). After mitosis, each daughter cell inherits a centriole/pro-centriole pair that docks on the membrane to form a basal body and to elongate an axoneme. (b) De novo duplication pathway in nondividing (G0) cells. The deuterosome (grey) produces multiple centrioles (red) that dock at the membrane to form basal bodies and to elongate an axoneme.
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An alternative pathway of basal body duplication is found in multiciliated animal cells, such as airway epithelial cells (Fig. 2b). This pathway allows for the rapid de novo formation of multiple centrioles around an electron-dense structure, termed the deuterosome (reviewed in Beisson & Wright, 2003; Bettencourt-Dias & Glover, 2007; Nigg & Stearns, 2011). Once formed, centrioles migrate to the cell membrane where they dock and act as basal bodies for axonemal elongation. An interesting feature of metazoa is that the de novo formation of centrioles is possible in most differentiated cell types, unless the process is actively suppressed by pre-existing centrioles (reviewed in Nigg & Stearns, 2011). The inhibitory effect of existing centrioles on de novo formation is further suggested by the observation that de novo assembly can occur in dividing cells if the centriole is removed (Khodjakov et al., 2002).
Although the templated and de novo pathways of duplication predominate in the metazoa, variant pathways have been observed in the sperm cells of the termite Mastotermes darwiniensis (Riparbelli et al., 2009) and in unfertilized eggs of some parthenogenetic insects (Riparbelli & Callaini, 2003). In these cases, centrioles are formed de novo without the need for deuterosomes or a template centriole. Notably, many of the regulatory components involved in the duplication process are conserved across species that use the templated, de novo and variant pathways (Azimzadeh & Marshall, 2010) and, as such, mechanistic differences may arise through the fine-tuning of a common set of key proteins.
Following centriole duplication, basal body maturation and axonemal elongation occur to form a cilium (Sorokin, 1962). Within animal epithelial cells, docking of the basal body is associated with the actin–myosin network and is mediated by the planar cell polarity (PCP) pathway. A key PCP protein, Dishevelled, has been shown to localize to the apical surface of multiciliated epithelial cells (Park et al., 2006) and to regulate both the docking and planar polarization of basal bodies in Xenopus laevis epidermal cells (Park et al., 2008). In most cases, mature basal bodies dock to the membrane and elongation then occurs through the delivery of ciliary components to the growing tip by a process of IFT (Kozminski et al., 1993; Parker & Katsanis, 2011). This transport is bidirectional: kinesin-2 mediates anterograde movement, whereas cytoplasmic dynein-2 performs retrograde movement (Cole et al., 1998; Pazour et al., 1999). In some cases, however, such as in Plasmodium yoelii (Sinden et al., 1976) and in Drosophila melanogaster sperm cells (Phillips, 1970), elongation occurs in the cytoplasm in an IFT-independent manner, and membrane docking occurs after axonemal elongation. In Plasmodium, cilia always form in the cytoplasm and IFT genes have been lost from the genome (Briggs et al., 2004). By contrast, the Drosophila genome possesses the IFT gene cohort, and all cell types apart from sperm cells utilize the IFT elongation mechanism (Han et al., 2003; Sarpal et al., 2003).
3. Diversity of cilia form and function
In animal cells, cilia have distinct nonmotile and motile forms. In general, motile cilia possess axonemal dynein arms that generate sliding force and hence the ciliary beat, and also usually contain a central microtubule pair (reviewed in Satir & Christensen, 2007). In contrast, nonmotile cilia lack both the central apparatus and dynein motors associated with beating and, instead, carry out a sensory role (reviewed in Singla & Reiter, 2006). Because examples of motile cilia are found in extant species of all the major eukaryotic lineages, and overall cilia morphology is highly conserved both within and between lineages, it is widely believed that the LECA also possessed a motile cilium (Cavalier-Smith, 1978; Luck, 1984; reviewed in Satir & Christensen, 2007). Based on comparative analyses of ciliary structure and function across a range of eukaryotes, it is further hypothesized that cilia of the LECA could perform both motility and sensory functions (Cavalier-Smith, 1978; Hodges et al., 2010; Pereira-Leal et al., 2010; Carvalho-Santos et al., 2011; Wickstead & Gull, 2011). Notably, the restricted distribution of immotile cilia within specific phylogenetic clades, such as in the evolutionary distant metazoa and centric diatoms (Jensen et al., 2003), implies that nonmotile cilia evolved from motile ancestors on independent occasions. Despite this apparent parallel evolution, nonmotile cilia display a remarkable conservation of structure, possessing a ‘9 + v’ (variable) microtubule arrangement (reviewed in Gluenz et al., 2010).
In addition to the structural differences that are clearly associated with motile vs nonmotile ciliary function, other structural variations are apparent, even within functionally similar cilia. Such variation can be seen in the nematode Caenorhabditis elegans, where immotile sensory cilia lack a canonical triplet basal body structure (Perkins et al., 1986). Other examples include the addition of nine outer dense fibres surrounded by a fibrous sheath in mammalian sperm tails (Fawcett, 1975; Eddy et al., 2003), the para-axonemal rods of Giardia (Holberton, 1973) and the elaborate paraflagellar rod structure found in species such as the kinetoplastid protozoa (Bastin et al., 1996).
4. Diversity of ciliomes
Consistent with the observed variations in ciliary form and function, there is considerable variation in the protein composition of cilia (the ‘ciliome’) in different species. Over the years, the characterization of genes encoding ciliary proteins has been facilitated by a number of experimental approaches, with mutant screens and protein purification being two of the most established (Lewin, 1952, 1953; Brokaw et al., 1982; Brokaw & Luck, 1983). Examples of genes identified in this way include uniflagellate-1 (Brokaw et al., 1982) and fla10 (Walther et al., 1994) in Chlamydomonas (mutational studies reviewed in Dutcher, 1989), and the ciliary adenosine triphosphatase isolated from Tetrahymena (Gibbons, 1963). For many years, cross-species comparisons of genes such as these were limited by the extent to which gene sequences were conserved, because experiments often relied on DNA hybridization assays.
In another form of ‘mutant’ analysis, the study of ciliopathies (human diseases caused by defects in cilia) has identified ciliary proteins that can have tissue- and even cell type-specific roles (Fliegauf et al., 2007). For example, mutations in genes encoding polycystin 1/2 proteins (PKD1/2) perturb ciliary function in kidneys, leading to polycystic kidney disease, yet cilia in the trachea and in sperm are normal (Yoder et al., 2002). Similarly, Kartagener’s syndrome is the result of reduced ciliary beat in the respiratory tract and sperm cells as a result of defects in inner/outer dynein arms (reviewed in Chodhari et al., 2004). Such examples imply either that differences in cilia phenotypes result from variation in the role of ciliary proteins between cell types, or that some cell types may be more resilient to mutations in ciliary components than others.
Although mutant and biochemical analyses of individual proteins have been hugely important to our understanding of the cilium, the recent application of proteomic methods to the study of cilia has allowed catalogs of ciliary proteins to be compared between species and at great evolutionary distance (Keller et al., 2005, 2006; Pazour et al., 2005; Broadhead et al., 2006; Kilburn et al., 2007). Direct comparison between these ciliomes can identify proteins shared between sets, but can also be used to test whether ciliary proteins from one organism are encoded in the genome of another. Both of these types of comparison have revealed that, although the ciliary form is extensively conserved, there is a surprising lack of homology between the components of different cilia. For example, of 331 proteins identified as part of the ciliary matrix of the protist Trypanosoma brucei (Broadhead et al., 2006), only 49 orthologues were identified in a similar preparation from the alga Chlamydomonas reinhardtii (Pazour et al., 2005).
From the pool of ciliary proteins that are present in more than one species, bioinformatic analyses have facilitated the identification of genes that encode a core cohort that is shared across all extant eukaryotes (e.g. Avidor-Reiss et al., 2004; Broadhead et al., 2006; Merchant et al., 2007). The phylogenetic distribution of genes encoding these core proteins implies that many were present in the LECA (Hodges et al., 2010; Pereira-Leal et al., 2010). These core ciliary proteins include some (such as tubulins) that have important cellular functions outside of their ciliary role, and others that have become highly specialized for their ciliary role (e.g. inner- and outer-arm dyneins, radial spoke proteins) (Piperno et al., 1977; Gibbons, 1995; Kamiya, 1995; reviewed in Satir, 1998 and King, 2000).
Although the core protein cohort reveals conservation in cilia structure and function, and can be used to infer some of the likely properties of the LECA, differences between ciliomes are a reflection of evolutionary divergence. Ciliome divergence can be assessed in two complementary ways. In the first, genomes from a wide range of species can be scanned for the presence or absence of genes encoding proteins known to have a ciliary role in at least one of those species. For example, it has been shown that genes encoding the basal body proteins VFL1, SAS-4 and SAS-6, and also each of the axonemal dynein classes, are not ubiquitous to all ciliated species (Silflow et al., 2001; Pfannenschmid et al., 2003; Delattre et al., 2006; Nakazawa et al., 2007; Wickstead & Gull, 2007; Dammermann et al., 2008; Hodges et al., 2010). Furthermore, the absence of the triplet basal body structure in cilia of Caenorhabditis elegans has been shown to be associated with the loss of a cohort of genes encoding typical ciliary proteins (Perkins et al., 1986; Hodges et al., 2010; Pereira-Leal et al., 2010). In the second approach, novel ciliary components can be identified through trait map comparisons of genomes of ciliated vs nonciliated organisms (Hodges et al., 2010, 2011). In this way, 213 orthologues have been identified that each possess a phylogenetic distribution suggestive of conserved ciliary function. Many of these genes have not previously been associated with ciliogenesis.
5. Ciliary loss
Loss of cilia has occurred on multiple independent occasions during the evolution of eukaryotes, perhaps most notably in fungi and land plants (reviewed in Renzaglia & Garbary, 2001; Liu et al., 2006). In the fungi, loss most probably occurred just once prior to the divergence of the ascomycetes, basidiomycetes and zygomycetes (Liu et al., 2006). Given that cilia evolution is marked by both conservation and diversification, and that whole-genome analysis can identify associated changes in genome composition, it is now possible to associate changes in genome composition with the loss of the ability to form cilia.